The RNA polymerase of Escherichia coli is a large, multisubunit enzyme existing in two forms. The core enzyme, consisting of subunits xcex2 and xcex2xe2x80x2 and an a subunit dimer, carries out processive transcription elongation followed by termination (Helmann et al., 1988). When one of a variety of sigma ("sgr") factors is added to core, the holoenzyme is formed (Burgess et al., 1969). The "sgr" subunit confers promoter-specific DNA binding and transcription initiation capabilities to the enzyme (Helmann et al., 1988; Burgess et al., 1969; Gross et al., 1996; Gross et al., 1992). "sgr"70 of E. coli was the first a factor to be described and characterized (Burgess et al., 1969). Since then, numerous a factors have been discovered throughout the Eubacterial kingdom, including six alternative a factors in E. coli. Each "sgr" subunit directs its cognate holoenzyme to start transcription from only those promoters containing DNA sequences specifically recognized by the "sgr" factor. Thus, generally, each a directs transcription initiation from a specific set of promoters to transcribe genes with related functions. This control of transcription is mediated partially through the competition of the individual "sgr" factors for the core enzyme and is a major part of global gene regulation in bacteria (Zhou et al., 1992).
As the number of identified "sgr" factors increased, it became apparent that they shared several regions of amino acid sequence similarity (Helmann et al., 1988; Gribskov et al., 1986; Lonetto et al., 1992), and the function of the conserved regions is of continuing interest (Waldburger et al., 1994; Dombrowski et al., 1993; Siegele et al., 1989; Gardella et al., 1989; Lesley et al., 1989). Deletion analysis of "sgr"70 identified a segment of the protein that overlaps conserved region 2.1 (residues 361-390) as being necessary and sufficient for core binding (Lesley et al., 1989). A mutation in a homologous region of Bacillus subtilis "sgr"E has also been shown to affect core binding (Shuler et al., 1995). However, recent findings of core binding mutations in other conserved and nonconserved regions of "sgr"32 have led to the idea of multiple binding sites for the "sgr" subunit on the core enzyme (Joo et al., 1997; Zhou et al., 1992; Joo et al., 1998; Sharp et al., 1999).
The xcex2 and xcex2xe2x80x2 subunits each contain regions that have high sequence homology with the two largest subunits of eukaryal polymerases (Allison et al., 1985; Sweetser et al., 1987; Jokerst et al., 1989). Some of these conserved regions may act as interaction domains. An interaction domain is the minimal region of a protein that can independently fold to form the secondary and tertiary structure required to interact with another protein, DNA, RNA, or ligand. Interaction domains are larger than the actual binding site which is formed by the amino acids in direct contact with the binding partner. Severinov et al. (Severinov et al., 1992, 1995 and 1996) demonstrated the domain-like properties of xcex2 and xcex2xe2x80x2 by reconstitution of functional RNA polymerase from fragmented xcex2 and xcex2xe2x80x2 subunits. Thus, the properties of the polymerase do not require the entire intact length of the subunit but rather can be generated with smaller domain modules.
There have been two observations that have identified deletions in the xcex2 or xcex2xe2x80x2 subunits that produce subunits still capable of forming core enzyme structures but not the holoenzyme. First, a xcex2 subunit truncation, missing approximately 200 amino acids of the C terminus, was shown by glycerol gradient centrifugation to migrate with the other core subunits but was never seen in the "sgr"-containing fractions (Glass et al., 1986). Second, when immunoprecipitation assays were performed using reconstituted RNA polymerase containing xcex2xe2x80x2 deletion mutants missing amino acids 201-477, the core subunits were recovered in the same fraction but lacked "sgr" (Luo et al., 1996). However, it was unclear whether the xcex2xe2x80x2 deletion was non-specific, e.g., prevented correct formation of the interaction domain.
The idea that "sgr" binding is affected by perturbations of the C terminus of xcex2 and the N terminus of xcex2xe2x80x2 is consistent with experiments showing that these two subunit termini are physically close together and can be fused through a flexible linker and still form a functional enzyme (Severinov et al., 1997). Recent protein-protein footprinting data have identified a similar region on xcex2xe2x80x2 and two new sites on xcex2 for possible interactions with the "sgr"70 subunit (Owens et al., 1998). While Owens et al. showed that residues 228-461 of xcex2xe2x80x2 are physically close to "sgr", the authors did not conclude that there is a direct interaction between xcex2xe2x80x2 and "sgr".
Burgess et al. (1998) report that residues 260 to 309 of xcex2xe2x80x2 bind to a based on the use of in vitro far-Western and co-immobilization assays. However, in vitro cell-free binding results do not evidence that the region involved in binding in vitro is involved in binding in vivo. For example, it is possible that this region of xcex2xe2x80x2 is buried in the native structure, e.g., a hydrophobic region, and so would not play a role in vivo binding. Structural analysis programs indicate that xcex2xe2x80x2260-309 has two xcex1 helices joined by a random coil, and that these two helices are amphipathic and have the potential for coiled coil formation, based on a heptad repeat motif (Chao et al., 1998; Cohen et al., 1986; Lupas et al., 1991). In particular certain positions known as a and d in the coiled coil motif are hydrophobic and so may be buried in native xcex2xe2x80x2
Thus, what is needed is the identification of a region in the subunits of core RNA polymerase that interacts with "sgr" in vivo. What is also needed is a method to identify specific inhibitors of the binding of "sgr" to core RNA polymerase.
The invention provides an isolated and purified xcex2xe2x80x2 subunit of RNA polymerase or a portion (i.e., fragment) thereof which specifically binds to "sgr" in vivo. Preferably, the portion comprises at least 39, more preferably at least 44, and even more preferably at least 49, residues of the xcex2xe2x80x2 subunit, although smaller fragments which specifically bind to "sgr" in vivo are also envisioned. Also preferably, the isolated and purified portion of the xcex2xe2x80x2 subunit comprises residues 270 to 309, and even more preferably residues 260 to 309. As described hereinbelow, a region on the xcex2xe2x80x2 subunit of RNA polymerase was identified that interacts directly with "sgr" (the interaction domain). The in vitro interaction domain of the xcex2xe2x80x2 subunit with "sgr" was identified by far-Western blot analysis, which is a general method for mapping a domain on one protein that is necessary for binding another protein, and a co-immobilization assay. As used herein, an xe2x80x9cinteraction domainxe2x80x9d refers to the minimal region of a protein that can independently fold to form the secondary and tertiary structures required to interact with another protein, DNA, RNA or ligand. The "sgr" binding region of xcex2xe2x80x2 was found to interact with various a factors, including "sgr"70 and several other E. coli "sgr"""s, T4 phage "sgr" gp 55, and "sgr"A from Bacillus subtilis. 
As also described hereinbelow, proteins were prepared which had single point mutations in the predicted coiled coils located within residues 260-309 of xcex2xe2x80x2. Several of the mutants were defective for binding "sgr"70 in vitro. Of these mutants, three (R275Q, E295K, and A302D which are change-of-charge mutants at the e and g residues of the xcex2xe2x80x2260-309 predicted coiled coil) were completely defective for growth in an in vivo assay where the mutant xcex2xe2x80x2 is the sole source of xcex2xe2x80x2 subunit. All of the mutants were able to assemble into the core enzyme, however, R275Q, E295K, and A302D were defective for E"sgr"70-holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor "sgr" factors. Some mutations were nonfunctional in some of the assays but functional in others, indicating that binding of other sites may compensate for loss of binding at the xcex2xe2x80x2260-309 site. Thus, these results showed that residues 260 to 309 of the xcex2xe2x80x2 subunit specifically bind to "sgr" in vivo, and that mutations in this region can greatly diminish core binding of "sgr"70 and other minor "sgr"""s. In the recently published crystal structure of Thennus aquaticus core RNA polymerase (Zhang et al., 1999), the region homologous to xcex2xe2x80x2260-309 of E. coli forms a coiled coil. Modeling of the xcex2xe2x80x2 mutations described herein onto that coiled coil places the most defective mutations on one face of the helix, which may indicate where most of the contact surface with "sgr"70 occurs. As RNA polymerase is a large multi-subunit complex (having about 3300 amino acids), and the xcex2xe2x80x2 subunit of RNA polymerase is a large protein, e.g., the xcex2xe2x80x2 subunit of E. coli is about 155,000 daltons, the identification of the region of the core RNA polymerase which specifically interacts with "sgr" in vivo represents a significant finding as it provides a specific target for drug discovery, e.g., drugs which specifically interfere with the core-"sgr" interaction.
Thus, the invention provides a method to identify an agent which inhibits or prevents the binding of "sgr" to core RNA polymerase, a subunit thereof or a portion of the subunit. The method comprises contacting the agent with core RNA polymerase, e.g., isolated core RNA polymerase, or an isolated subunit of RNA polymerase or a portion thereof so as to form a complex. As used herein, xe2x80x9cisolated and/or purifiedxe2x80x9d refers to in vitro preparation, isolation and/or purification of a protein or a complex of biomolecules, e.g., core RNA polymerase, so that it is not associated with in vivo substances or is substantially purified from in vitro substances. Preferably, the portion of the subunit comprises at least 39 amino acids, more preferably at least 44 amino acids, even more preferably at least 49 amino acids, of the xcex2xe2x80x2 subunit. The complex is then contacted with "sgr" or a portion thereof and it is determined whether the agent inhibits or prevents the binding of "sgr" to core RNA polymerase, the isolated subunit of RNA polymerase or portion thereof. A portion of a comprises at least 30, preferably at least 55, more preferably at least 100, and even more preferably at least 140 residues of "sgr", although smaller fragments which specifically bind to xcex2xe2x80x2 in vivo are also envisioned. Alternatively, the agent is contacted with the core RNA polymerase, a subunit and or portion thereof and "sgr" or a portion thereof, i.e., simultaneously. The "sgr" may be a homologous "sgr", for example, if the core RNA polymerase or the isolated subunit of RNA polymerase is that of E. coli, the "sgr" is a "sgr" which is encoded by the genome of E. coli. Alternatively, the "sgr" may be a heterologous "sgr", e.g., a phage-encoded "sgr".
Further provided is a method which comprises contacting the agent with isolated "sgr" or a portion thereof so as to form a complex. The complex is then contacted with isolated core RNA polymerase, or an isolated subunit of RNA polymerase or a portion thereof and it is determined whether the agent inhibits or prevents the binding of "sgr" to core RNA polymerase, the isolated subunit of RNA polymerase or portion thereof.
To find new inhibitors of bacterial transcription, a homogenous luminescence resonance energy transfer (LRET) based assay was developed on the basis of the fluorescent-labeled proteins "sgr"70 and xcex2xe2x80x2-fragment (residues 100-309). For the assay, "sgr"70 was labeled with a europium chelate (Eu(III)-DTPA-AMCA-maleimide) as the LRET donor and xcex2xe2x80x2 was labeled with IC5-maleimide as the acceptor. Measuring time-resolved fluorescence with the labeled proteins permitted the monitoring of binding of "sgr"70 to xcex2xe2x80x2 by observing the emission of the LRET acceptor (IC5-labeled xcex2xe2x80x2-fragment). The emission of the acceptor is sensitized by an energy transfer from the LRET donor (DTPA-AMCA-Eu-complex-labeled "sgr"70) that occurs when the dyes come into close proximity to each other ( less than 75 xc3x85). Due to its naturally short lifetime of several nanoseconds, the residual IC5-fluorescence acquired after 50 microseconds is due solely to LRET, reducing the background signal to a minimum and hence providing a good signal-to-noise ratio. The assay was used to measure the effect of the environment (solvents, denaturants, and salt) and can be used to measure the effect of potential inhibitors on the binding of "sgr"70 to the xcex2xe2x80x2-fragment. Such an assay is particularly well suited for high-throughput screening.
Also provided is a method to identify a region on a subunit of core RNA polymerase which specifically binds "sgr". The method comprises contacting core RNA polymerase, e.g., isolated core RNA polymerase, an isolated subunit thereof or a portion thereof with "sgr" or a portion thereof so as to form a complex. The core RNA polymerase, isolated subunit or portion thereof comprises at least one amino acid substitution. Then complex formation is detected or determined and, for example, compared to complex formation between core RNA polymerase, an isolated subunit or portion thereof, which does not comprise an amino acid substitution, and "sgr" or a portion thereof.
The invention further provides a method to identify an agent which inhibits or prevents the binding of "sgr" to the xcex2xe2x80x2 subunit of core RNA polymerase. The method comprises contacting a prokaryotic cell with the agent and detecting or determining whether the agent inhibits or prevents the binding of "sgr" to the xcex2xe2x80x2 subunit of RNA polymerase in the cell. The cell may be a recombinant cell, i.e., a cell which is augmented by exogenously introduced nucleic acid, e.g., by transformation or transduction. Thus, the invention also provides a host cell comprising a recombinant DNA encoding a xcex2xe2x80x2 subunit of RNA polymerase.
The invention further provides agents identified by the methods of the invention and, in particular, agents which inhibit the growth of prokaryotic cells which are associated with disease, see e.g., Zinsser Microbiology (17th ed., Appleton-Century-Crofts, NY (1980).